Mak D O, McBride S, Raghuram V, Yue Y, Joseph S K, Foskett J K
Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Gen Physiol. 2000 Mar;115(3):241-56. doi: 10.1085/jgp.115.3.241.
The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an intracellular Ca(2+)-release channel localized in endoplasmic reticulum (ER) with a central role in complex Ca(2+) signaling in most cell types. A family of InsP(3)Rs encoded by several genes has been identified with different primary sequences, subcellular locations, variable ratios of expression, and heteromultimer formation. This diversity suggests that cells require distinct InsP(3)Rs, but the functional correlates of this diversity are largely unknown. Lacking are single-channel recordings of the recombinant type 3 receptor (InsP(3)R-3), a widely expressed isoform also implicated in plasma membrane Ca(2+) influx and apoptosis. Here, we describe functional expression and single-channel recording of recombinant rat InsP(3)R-3 in its native membrane environment. The approach we describe suggests a novel strategy for expression and recording of recombinant ER-localized ion channels in the ER membrane. Ion permeation and channel gating properties of the rat InsP(3)R-3 are strikingly similar to those of Xenopus type 1 InsP(3)R in the same membrane. Using two different two-electrode voltage clamp protocols to examine calcium store-operated calcium influx, no difference in the magnitude of calcium influx was observed in oocytes injected with rat InsP(3)R-3 cRNA compared with control oocytes. Our results suggest that if cellular expression of multiple InsP(3)R isoforms is a mechanism to modify the temporal and spatial features of Ca(2+) signals, then it must be achieved by isoform-specific regulation or localization of various types of InsP(3)Rs that have relatively similar Ca(2+) permeation properties.
肌醇1,4,5 - 三磷酸受体（InsP(3)R）是一种位于内质网（ER）的细胞内钙释放通道，在大多数细胞类型的复杂钙信号传导中起核心作用。已鉴定出由多个基因编码的InsP(3)R家族，它们具有不同的一级序列、亚细胞定位、可变的表达比例以及异源多聚体形成。这种多样性表明细胞需要不同的InsP(3)R，但这种多样性的功能相关性在很大程度上尚不清楚。目前缺乏对重组3型受体（InsP(3)R - 3）的单通道记录，该受体是一种广泛表达的亚型，也与质膜钙内流和细胞凋亡有关。在这里，我们描述了重组大鼠InsP(3)R - 3在其天然膜环境中的功能表达和单通道记录。我们所描述的方法为在内质网膜中表达和记录重组内质网定位离子通道提出了一种新策略。大鼠InsP(3)R - 3的离子通透和通道门控特性与同一膜中的非洲爪蟾1型InsP(3)R惊人地相似。使用两种不同的双电极电压钳方案来检测钙库操纵的钙内流，与对照卵母细胞相比，注射大鼠InsP(3)R - 3 cRNA的卵母细胞在钙内流幅度上没有差异。我们的结果表明，如果多种InsP(3)R亚型的细胞表达是一种改变[Ca(2+)]i信号时空特征的机制，那么它必须通过具有相对相似钙通透特性的各种类型InsP(3)R的亚型特异性调节或定位来实现。
